Methods for detecting antibodies in mucosal samples and device for sampling mucosal material

ABSTRACT

A method to detect local antibodies such as antigen-specific IgE via a brush biopsy specimen of a mucosal surface of a subject is disclosed. The method is easily performed in an office setting on both adult and pediatric patients. Also disclosed is a brush device specially designed for harvesting materials from a mucosal surface such as the medial surface of the inferior turbinate.

BACKGROUND OF THE DISCLOSURE

Since the early 1970s, it has been known that antigen-specific IgE canbe recovered from nasal and bronchial secretions in allergic patientswho are challenged with that antigen (Yoshida T, 2005). It has also beendemonstrated that IgE can be present in nasal secretions even thoughskin testing (Houri M, 1972) and in vitro testing (Stenius B, 1971) arenegative, and it has been suggested that this locally produced IgE cancause an inflammatory reaction in a specific part of the airway, withoutaffecting the rest of the body. In 1985, Ohashi et al. demonstratedelevated IgE levels in homogenized nasal mucosa from twelve patientswith symptoms suggestive of allergy who were negative on skin and RASTtesting (Ohashi Y, 1985).

Local IgE production has also been recognized in the pathogenesis ofsuch conditions as allergic fungal rhinosinusitis (Pant H, 2009) andnasal polyposis (Sabirov A, 2008). Levels of Alternaria-specific IgE innasal polyp tissue have been found to be significantly higher thannon-polyp tissue from patients with chronic rhinosinusitis, and 60% ofpatients with nasal polyps had evidence of local IgE without evidence ofAlternaria-specific IgE in the serum (Sabirov A, 2008). In addition,local IgE may also be responsible for symptoms in patients with chronicrhinitis who are diagnosed with “non-allergic rhinitis” based solely onnegative skin and blood testing (Durham S R, 2000).

Current methods to collect local IgE in the nose include nasal lavage(Yoshida T, 2005) and surgical biopsy of nasal tissues (Ahn C N, 2009).However, nasal lavage is a difficult test to perform in the officesetting, particularly in the pediatric population. In addition, it isuncomfortable for the patient and has a high rate of technicalvariability, which limits the interpretation of the results. Nasallavage specimens can only collect IgE that is present in the mucus, butthere is additional IgE present in the epithelium of the inferiorturbinates (Ahn C N, 2009). Surgical biopsy will detect epithelial IgE,but this is an invasive test which is not practical in the officesetting or ethical to perform solely for the purpose of allergy testing.

Brush biopsy with a cytology brush has been used inside the nose for thestudy of ciliary ultrastructure (Rutland J, 1982) and for viral culture(Winther B, 1986), but has not been used for collecting mucosal materialand antibody testing.

SUMMARY OF THE DISCLOSURE

In one aspect, the instant disclosure is directed to a method ofdetecting local antibody molecules by obtaining a brush biopsy samplefrom a mucosal surface of a subject, and determining the presence ofantibodies in the sample. In a specific embodiment, a brush biopsyspecimen of the inferior turbinate is collected, which can be easilyperformed in the office setting on both adult and pediatric patients.Total IgEs or antigen-specific IgE can be detected in a collected brushbiopsy specimen using standard methods.

In another embodiment, the instant disclosure provides a device designedfor collecting a brush biopsy sample from a mucosal surface of asubject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A specific embodiment of the device disclosed herein forretrieving a brush biopsy sample from medial surface of the inferiorturbinate.

FIG. 2. Anatomy of the nose.

DETAILED DESCRIPTION OF THE DISCLOSURE

In the following description, reference is made to the accompanyingdrawings that form a part hereof, and in which is shown by way ofillustration specific embodiments which may be practiced. Theseembodiments are described in detail to enable those skilled in the artto practice the invention, and it is to be understood that otherembodiments may be utilized and that structural, logical and electricalchanges may be made without departing from the scope of the presentinvention. The following description of example embodiments is,therefore, not to be taken in a limited sense.

The Abstract is provided to comply with 37 C.F.R. § 1.72(b) to allow thereader to quickly ascertain the nature and gist of the technicaldisclosure. The Abstract is submitted with the understanding that itwill not be used to interpret or limit the scope or meaning of theclaims.

It has been recognized in accordance with the instant disclosure that abrush biopsy sample can be effectively retrieved from a mucosal surface(such as the inferior nasal concha) for detecting local antibodymolecules, and the antibody molecules detected can be used in diagnosisof various conditions and disorders. Accordingly, this disclosureprovides methods for detecting local antibodies in a brush biopsy sampleas well as devices for retrieving a brush biopsy sample.

In one aspect, the instant disclosure is directed to a method ofdetecting local antibody molecules by obtaining a brush biopsy samplefrom a mucosal surface of a subject, and determining the presence ofantibodies in the sample.

By a “mucosa,” it is meant a membrane that constitutes the surfacelining of body cavities/orifices, respiratory and gastrointestinaltracts, internal organs and other body parts, and is typically coveredwith epithelial cells. An orifice is an opening of a body and includes,for example, the ear, the nose, the mouth, the urethral orifice, and theanus. Thus, a mucosal surface can be, for example, the inner surface ofthe nose, mouth, throat, esophagus, the stomach, the small intestine,the large intestine, the rectum, the urethra, vagina, uterus, or cervix,as well as the surface of the tongue or adenoids, the prepuce, andglans.

By a “subject,” it is meant an individual mammal. In some embodiments,the mammal is a human, a primate, a domestic cat, a domestic dog, a cow,a sheep, a goat, a pig, a rat, or a mouse.

The term “local antibodies” is used to refer antibodies present incertain tissues or parts of a body that do not necessarily circulatethrough the blood and spread to other tissues or parts of the body, asopposed to antibodies present in the blood (or “systemic antibodies”).

As disclosed herein, to achieve detection of local antibodies at amucosal surface, a brush biopsy sample is obtained from the mucosalsurface. For example, a biopsy brush is inserted into an orifice of asubject so that the biopsy brush contacts a mucosal surface in theorifice, and the brush is manipulated, e.g., being applied with anappropriate amount of pressure or rotated against the mucosal surface,so that sufficient mucosal materials becomes associated with the biopsybrush, and removed from the orifice of the subject.

It has been established in this disclosure that adequate mucosalmaterial can be obtained by brush biopsy, which is believed to containboth mucosal secretions and cellular materials including epithelialcells of the mucosa. As disclosed herein, such brush biopsy sample canbe released from the brush (e.g., by washing or immersing the brush withsuitable solutions such as a saline solution), and then used indetecting the presence and/or amount of antibody molecules(immunoglobulins) in the sample.

Detection can be directed to the entire population of immunoglobulins inthe sample, or to immunoglobulins of particular isotypes. In someembodiments, detection is directed to the IgA, IgD, IgE, IgG, and IgM,which are the immunoglobulin isotypes in humans. In other embodimentswhere the subject being tested is a mammal other than human, detectionis directed to immunoglobulin isotypes of other mammals. In someembodiments, the immunoglobulins are of particular immunoglobulinisotype subclasses, for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2,which are the immunoglobulin isotypes in humans. In other embodiments,the immunoglobulin isotype classes are the isotype classes of othermammals.

In some embodiments, total antibody molecules in a biopsy sample aredetected irrespective antigen specificity. In other embodiments,antigen-specific antibody molecules of all types (i.e., all isotypes andsubclasses) or of particular isotype or subclass are being detected.

In specific embodiments, the antigen is an allergen. By “allergen,” itis meant a molecule that is capable of inducing an allergic response ina subject. The detection method disclosed herein is capable of detectingantibodies against various allergens. In some embodiments, the allergenderives from a plant (a “plant allergen”). In specific embodiments, theallergens derive from the White Oak tree (Quercus alba), Timothy Grass(Phleum pratense), ragweeds (Ambrosia spp., e.g., Ambrosiaartemisilfolia). A plant allergen can derive from various parts of aplant, for example, the pollen of the plant. In other embodiments, theallergen is a microbial allergen, such as Alternaria spp. (e.g.,Alternaria alternata), and Aspergillus spp. (e.g., Aspergillusfumigata). In some other embodiments, the allergens derive from animals,such as domestic cat, dog, house dust mites (Dermatophagoides spp.), andcockroaches (animals of the order Blattaria). In additional embodiments,the allergen derives from food, e.g., milk, eggs, peanuts, tree nuts,seafood, shellfish, soy, rice, and wheat.

In other embodiments, the antigens can be specific molecules, such asDerP1, FelD1 and the like.

Antibodies, both total antibodies and antigen-specific antibodies, canbe detected using assays and techniques known in the art. Suitableassays include direct immunofluorescence, indirect immunofluorescence,radioimmunoassay, enzyme-linked immunosorbent assay (ELISA),immunoprecipitation, Western blotting, isoelectric focusing,immunoelectrophoresis, radial immunodiffusion, flow cytometry, surfaceplasmon resonance, or bioassay. In some embodiments, the assay is acapture or “sandwich” ELISA. In other embodiments, the assay is anImmunoCAP® assay. The basis of the ImmunoCAP® technology is a 3dimensional cellulose polymer in a plastic reserve. This provides highbinding capacity of allergen proteins, including those present in verylow levels, providing increased sensitivity, specificity, andreproducibility. The commercial source of this immunofluorescence assayis Phadia (Portage, Mich.).

In an exemplary embodiment, a method is disclosed that effectivelydetects local IgE in nasal mucosa. More specifically, a brush biopsysample is retrieved from the inferior turbinate, also known as inferiornasal concha (see FIG. 2), and the sample is used to detect IgE, eithertotal IgE or antigen-specific IgE. The level of IgE detected can becompared to a reference value as a basis of diagnosis (e.g., diagnosisof allergy). IgE testing using brush biopsy samples has been foundherein to generally correlate with results from skin prick testing.Prior to this disclosure, brush biopsy has not been proposed forcollecting mucosal material and detecting local IgEs in connection withdiagnosis of allergies.

In a further aspect, the instant disclosure provides a device designedfor collecting a brush biopsy sample from a mucosal surface of asubject.

One device disclosed herein is named The RABBIT (Rotating AutomaticBrush Biopsy of the Inferior Turbinate), an exemplary configuration ofwhich is depicted in FIG. 1. Such device provides an easy, quick,inexpensive and non-invasive method for brush biopsy of the nose.Standard cytology brushes, which are used most commonly for cervicalbiopsy, are too long for safe, day to day clinical use in the nose. Theyoften contain metal components which may damage delicate tissues in thenose and are difficult to use quickly enough in the pediatric populationto obtain an adequate sample of epithelial cells.

The device (8) is designed to fit comfortably in the hand and weighsless than 1 pound. The shell of the device (9), preferably plastic, isapproximately 16×5×2 cm in dimension, and houses a motor or motors formoving the shaft and brush (4). The motor or motors may be powered by anelectric cord, and/or by a battery that may also be inside the shell;the battery may be rechargeable. The motor or motors may be turned onand off by a switch (7) or switches. The motor or motors may cause theshaft and brush to rotate, they may also cause the shaft and brush tomove axially. The switch (7) or switches may also control the axialmovement, or said axial movement may be automatic. The shaft and brush(4) enters the shell through a housing (5), and may be of a single piecewith the motor, in an embodiment in which the entire device is eitherintended for single use and disposed or is made to be sterilized, or maybe removable. If the shaft and brush are removable, a quick releaseswitch (6) or other actuator for quick switching may be located on theoutside of the shell. The shaft and brush (4) may be approximately 7 cmin length, and may be made of any suitably stiff yet pliable materialsuch as plastic. In a preferred embodiment, the shaft terminates with anatraumatic structure (1) such as a rubber ring or non-latex foam tip. Ashorter shaft/brush may be employed for pediatric patients. The shaftmay have markings (3) indicating the maximum depth to which the brushshould be inserted into the nose. The brushing elements (2) are disposedon the shaft for a length of 1-2 cm, and may extend 1-2 mm radiallyoutward from the shaft. Brushing elements (2) may be bristles, (as shownin FIG. 1) loops, ridges, corrugations, or alternatively, any scrapingcollection device. Brushing elements may be made from known material andfixedly attached to the surface of the shaft or, alternatively, they maybe inherently formed on the surface of the shaft.

Modifications to the device shown in FIG. 1 can be made to adapt thedevice for retrieving biopsy samples from a particular mucosal surface.For example, the length and material of the shaft can be selected tomake the brush move easily and flexibly through the GI tract andretrieve materials from a particular site in the GI tract.

The methods and the devices disclosed herein are useful for diagnosingdisease and conditions associated with or characterized by abnormallevels of antibodies, particularly abnormal levels of local antibodylevels. By “abnormal” it is meant that the level of antibodies in apatient having a disease or condition is significantly increased ordecreased as compared to normal, healthy subjects. Conditions suitablefor diagnosis in accordance with the methods and devices disclosedherein include allergy, allergic fungal rhinosinusitis, nasal polyposisor non-allergic rhinitis. The instant methods and devices areparticularly useful for diagnosing allergies, including seasonal andfood allergies, allergies to any allergens such as those describedabove. The methods and devices disclosed herein provide a convenientalternative to standard allergy testing in both the pediatric and adultpopulations.

EXAMPLES

The present description is further illustrated by the followingexamples, which should not be construed as limiting in any way. Thecontents of all cited references (including literature references,issued patents, and published patent applications as cited throughoutthis application) are hereby expressly incorporated by reference.

Brush biopsy was performed in the outpatient setting. After informedconsent was obtained, the nose was decongested and anesthetized withtopical 2% lidocaine and 1% phenylephrine. After 5 minutes, a standardcytology brush was inserted along the medial aspect of the anterior ⅓ ofthe inferior turbinate on the side of the more patent nostril. The brushwas rotated back and forth several times, while avoiding direct pressureon the septum.

After the brush was withdrawn, it was immersed in a 12×75 mm roundbottom polypropylene tube containing 1 cc of PBS. The brush was rotatedfor 1 minute and then irrigated with 1 cc of additional PBS to releaseall mucus and cells bound to the bristles, and the specimen was storedat −20° C. Prior to freezing, 0.5 cc of the PBS was set aside for BCAprotein assay to determine total protein level.

Specimens were sent to a reference laboratory (IBT Laboratories, Lenexa,Kans.) for ImmunoCAP® analysis to determine total IgE level as well asantigen-specific IgE levels for the nine selected antigens. Specimenswere standardized for total protein.

The population chosen for this study was approximately 20 humanpatients, 18 years of age or older, who demonstrated at least 1 positivereaction (wheal >3mm or 2 mm greater than the negative control) on skinprick testing using a battery of 9 inhalant allergens: White Oak,Timothy grass, Ragweed, Cat dander, Dog dander, Cockroach, Alternaria,Aspergillus, and Dermatophagoides farinae. Patients who hadimmunotherapy in the past were excluded from participation in thisstudy.

Nine of the twenty patients were subjected to a further study whichcompared brush biopsy of the inferior turbinate with skin prick testingfor the overall detection of antigen-specific IgE and the detection ofindividual antigens.

Each subject tested positive to one or more of the following antigens onskin prick testing (“SPT”): White Oak, Timothy Grass, Ragweed, Cat, Dog,Cockroach, Alternaria, Aspergillus (2 molds) and D. Farinae (house dustmite). Each patient underwent Brush Biopsy Testing (“BBT”) as describedabove. The brush biopsy samples obtained from the patients were analyzedby ImmunoCAP® and antigen-specific IgE responses were assessed. A brushbiopsy sample having an IgE level equal to or above 0.10 kU/L for anallergen was scored “positive” for that allergen whereas a brush biopsysample having an IgE level less than 0.10 kU/L for an allergen wasscored “negative” for that allergen. The “class” of response was alsodetermined based on the level of antigen-specific IgE detected. A higher“class” number indicates higher levels of IgE (Table 11).

TABLE 1 Results for Subject 1, a forty year-old female Flag/ ReferenceTest Result Class Units Ranges Immunoglobulin E (IgE) 6 IU/mL 5-79ImmunoCAP Cat Dander IgE 0.60 1 kU/L <0.35 Dog Dander IgE 0.21 0/1 kU/L<0.35 Timothy Grass <0.10 0 kU/L <0.35 (Phleum pratense) IgE RagweedShort/Common <0.10 0 kU/L <0.35 (Ambrosia artemisilfolia) IgE Oak White(Quercus alba) IgE 0.18 0/1 kU/L <0.35 Alternaria tenuis/alternata IgE<0.10 0 kU/L <0.35 Aspergillus fumigatus IgE <0.10 0 kU/L <0.35 Mite0.10 0/1 kU/L <0.35 Dermatophagoides farinae IgE Cockroach German 0.140/1 kU/L <0.35 (Blatella germanica) IgE

TABLE 2 Results for Subject 2, a thirty year-old female Flag/ ReferenceTest Result Class Units Ranges Immunoglobulin E (IgE) 12 IU/mL 4-59ImmunoCAP Cat Dander IgE 1.40 2 kU/L <0.35 Dog Dander IgE 1.15 2 kU/L<0.35 Timothy Grass <0.10 0 kU/L <0.35 (Phleum pratense) IgE RagweedShort/Common 0.11 0/1 kU/L <0.35 (Ambrosia artemisilfolia) IgE Oak White(Quercus alba) IgE 0.21 0/1 kU/L <0.35 Alternaria tenuis/alternata IgE0.11 0/1 kU/L <0.35 Aspergillus fumigatus IgE <0.10 0 kU/L <0.35 Mite0.12 0/1 kU/L <0.35 Dermatophagoides farinae IgE Cockroach German 0.140/1 kU/L <0.35 (Blatella germanica) IgE

TABLE 3 Results for Subject 3, a twenty-eight year-old male Flag/Reference Test Result Class Units Ranges Immunoglobulin E (IgE) 6 IU/mL4-59 ImmunoCAP Cat Dander IgE 0.20 0/1 kU/L <0.35 Dog Dander IgE 0.110/1 kU/L <0.35 Timothy Grass 0.18 0/1 kU/L <0.35 (Phleum pratense) IgERagweed Short/Common 0.47 1 kU/L <0.35 (Ambrosia artemisilfolia) IgE OakWhite (Quercus alba) IgE 0.21 0/1 kU/L <0.35 Alternaria tenuis/alternataIgE <0.10 0 kU/L <0.35 Aspergillus fumigatus IgE <0.10 0 kU/L <0.35 Mite0.10 0/1 kU/L <0.35 Dermatophagoides farinae IgE Cockroach German 0.130/1 kU/L <0.35 (Blatella germanica) IgE

TABLE 4 Results for Subject 4, a twenty-nine year-old female Flag/Reference Test Result Class Units Ranges Immunoglobulin E (IgE) 6 IU/mL4-59 ImmunoCAP Cat Dander IgE 0.23 0/1 kU/L <0.35 Dog Dander IgE 0.170/1 kU/L <0.35 Timothy Grass <0.10 0 kU/L <0.35 (Phleum pratense) IgERagweed Short/Common 0.11 0/1 kU/L <0.35 (Ambrosia artemisilfolia) IgEOak White (Quercus alba) IgE 0.16 0/1 kU/L <0.35 Alternariatenuis/altenata IgE 0.11 0/1 kU/L <0.35 Aspergillus fumigatus IgE <0.100 kU/L <0.35 Mite 0.11 0/1 kU/L <0.35 Dermatophagoides farinae IgECockroach German 0.14 0/1 kU/L <0.35 (Blatella germanica) IgE

TABLE 5 Results for Subject 5, a twenty-seven year-old male Flag/Reference Test Result Class Units Ranges Immunoglobulin E (IgE) 4 IU/mL4-59 ImmunoCAP Cat Dander IgE <0.10 0 kU/L <0.35 Dog Dander IgE <0.10 0kU/L <0.35 Timothy Grass <0.10 0 kU/L <0.35 (Phleum pratense) IgERagweed Short/Common <0.10 0 kU/L <0.35 (Ambrosia artemisilfolia) IgEOak White (Quercus alba) IgE 0.18 0/1 kU/L <0.35 Alternariatenuis/alternata IgE <0.10 0 kU/L <0.35 Aspergillus fumigatus IgE <0.100 kU/L <0.35 Mite 0.47 1 kU/L <0.35 Dermatophagoides farinae IgECockroach German 0.13 0/1 kU/L <0.35 (Blatella germanica) IgE

TABLE 6 Results for Subject 6, a twenty-five year-old male Flag/Reference Test Result Class Units Ranges Immunoglobulin E (IgE) 32 IU/mL4-59 ImmunoCAP Cat Dander IgE 2.73 2 kU/L <0.35 Dog Dander IgE 0.68 1kU/L <0.35 Timothy Grass 0.15 0/1 kU/L <0.35 (Phleum pratense) IgERagweed Short/Common <0.10 0 kU/L <0.35 (Ambrosia artemisilfolia) IgEOak White (Quercus alba) IgE 5.43 3 kU/L <0.35 Alternariatenuis/alternata IgE 0.13 0/1 kU/L <0.35 Aspergillus fumigatus IgE <0.100 kU/L <0.35 Mite 3.14 2 kU/L <0.35 Dermatophagoides farinae IgECockroach German 0.16 0/1 kU/L <0.35 (Blatella germanica) IgE

TABLE 7 Results for Subject 7, a thirty-six year-old male Flag/Reference Test Result Class Units Ranges Immunoglobulin E (IgE) 6 IU/mL5-79 ImmunoCAP Cat Dander IgE 0.10 0/1 kU/L <0.35 Dog Dander IgE 0.100/1 kU/L <0.35 Timothy Grass 1.13 2 kU/L <0.35 (Phleum pratense) IgERagweed Short/Common <0.10 0 kU/L <0.35 (Ambrosia artemisilfolia) IgEOak White (Quercus alba) IgE 0.18 0/1 kU/L <0.35 Alternariatenuis/alternata IgE <0.10 0 kU/L <0.35 Aspergillus fumigatus IgE 0.100/1 kU/L <0.35 Mite 0.12 0/1 kU/L <0.35 Dermatophagoides farinae IgECockroach German 0.13 0/1 kU/L <0.35 (Blatella germanica) IgE

TABLE 8 Results for Subject 8, a twenty-four year-old female Flag/Reference Test Result Class Units Ranges Immunoglobulin E (IgE) 18 IU/mL4-59 ImmunoCAP Cat Dander IgE 0.46 1 kU/L <0.35 Dog Dander IgE 0.44 1kU/L <0.35 Timothy Grass 1.57 2 kU/L <0.35 (Phleum pratense) IgE RagweedShort/Common 0.13 0/1 kU/L <0.35 (Ambrosia artemisilfolia) IgE Oak White(Quercus alba) IgE 2.12 2 kU/L <0.35 Alternaria tenuis/alternata IgE0.15 0/1 kU/L <0.35 Aspergillus fumigatus IgE 0.10 0/1 kU/L <0.35 Mite1.10 2 kU/L <0.35 Dermatophagoides farinae IgE Cockroach German 0.18 0/1kU/L <0.35 (Blatella germanica) IgE

TABLE 9 Results for Subject 9, a fifty-four year-old female Flag/Reference Test Result Class Units Ranges Immunoglobulin E (IgE) 3 IU/mL3-48 ImmunoCAP Cat Dander IgE 0.10 0/1 kU/L <0.35 Dog Dander IgE <0.10 0kU/L <0.35 Timothy Grass <0.10 0 kU/L <0.35 (Phleum pratense) IgERagweed Short/Common 0.12 0/1 kU/L <0.35 (Ambrosia artemisilfolia) IgEOak White (Quercus alba) IgE 0.17 0/1 kU/L <0.35 Alternariatenuis/alternata IgE <0.10 0 kU/L <0.35 Aspergillus fumigatus IgE <0.100 kU/L <0.35 Mite <0.10 0 kU/L <0.35 Dermatophagoides farinae IgECockroach German 0.15 0/1 kU/L <0.35 (Blatella germanica) IgE

TABLE 10 Comparison of Brush Biopsy Testing with Skin Prick Testing forsubjects 1-9. “Concordance” indicates the combined proportion ofallergens detected that were BBT⁺/SPT⁺ and BBT⁻/SPT⁻. BBT⁺/ BBT⁻/ BBT⁻/BBT⁺/ Subject SPT⁺ SPT⁻ SPT⁺ SPT⁻ Concordance 1 3 0 4 2 3/9 2 3 1 1 44/9 3 5 2 0 2 7/9 4 2 2 0 5 4/9 5 1 5 1 2 6/9 6 5 2 0 2 7/9 7 2 1 1 53/9 8 3 0 0 6 3/9 9 4 2 3 0 6/9 Total 28 15 10 28 43/81 Percentage34.60% 18.50% 12.30% 34.60% 53.10%

TABLE 11 Comparison of Brush Biopsy Testing with Skin Prick Testing byallergen tested. “Concordance” indicates the combined proportion ofallergens detected that were BBT⁺/SPT⁺ and BBT⁻/SPT⁻. BBT⁺/ BBT⁻/ BBT⁻/BBT⁻/ Antigen (IgE) SPT⁺ SPT⁻ SPT⁺ SPT⁻ Concordance White Oak 6 0 3 06/9 Timothy Grass 4 2 1 2 6/9 Ragweed 3 2 2 2 5/9 short/common CatDander 5 1 3 0 5/9 Dog 0 0 7 2 2/9 Cockroach 5 0 4 0 5/9 Alternaria 1 13 4 5/9 Aspergillus 1 2 1 5 6/9 D. Farinae 4 1 4 0 4/9

TABLE 12 Results of Brush Biopsy Testing by Level of Response (Class).Range of Total number of Class Responses (kU/L) patients in grade 0  0-0.1 25 0 or 1  0.1-0.35 41 1 (low) 0.35-0.7 6 2 (medium) 0.71-3.5 83 (high)  3.51-17.5 1 Total 81

TABLE 13 Total Number of Positive Tests. SPT BBT Number of 38 56positive tests

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1.-12. (canceled)
 13. A device for obtaining brush biopsy of theinferior turbinate, comprising a handle with at least one embedded motoroperably attached to a shaft to which a brushing element is attached.14. The device of claim 13, wherein said shaft is rotated by a motor topermit the brush element to brush against mucosal surface of theinferior turbinate while being rotated.
 15. The device of claim 13,wherein said shaft is moved axially by a motor.
 16. The device of claim13, wherein the mechanism for operably attaching the shaft to the motorfurther comprises a quick release mechanism.